Evaluation of mitogenome sequence concordance, heteroplasmy detection, and haplogrouping in a worldwide lineage study using the Precision ID mtDNA Whole Genome Panel

Highlights

• The Precision ID mtDNA Whole Genome Panel was evaluated in a large lineage study using different tissues.

• Samples included high quality reference samples and also challenging forensic samples.

• The vast majority of samples yielded full mitogenomes from diverse phylogenetic backgrounds.

• We observed very high concordance to previous Sanger and other MPS-based analyses.

• We observed differences in the display of heteroplasmy that deserve further research.

Abstract

The emergence of Massively Parallel Sequencing technologies enabled the analysis of full mitochondrial (mt)DNA sequences from forensically relevant samples that have, so far, only been typed in the control region or its hypervariable segments. In this study, we evaluated the performance of a commercially available multiplex-PCR-based assay, the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific), for the amplification and sequencing of the entire mitochondrial genome (mitogenome) from even degraded forensic specimens. For this purpose, more than 500 samples from 24 different populations were selected to cover the vast majority of established superhaplogroups. These are known to harbor different signature sequence motifs corresponding to their phylogenetic background that could have an effect on primer binding and, thus, could limit a broad application of this molecular genetic tool. The selected samples derived from various forensically relevant tissue sources and were DNA extracted using different methods. We evaluated sequence concordance and heteroplasmy detection and compared the findings to conventional Sanger sequencing as well as an orthogonal MPS platform. We discuss advantages and limitations of this approach with respect to forensic genetic workflow and analytical requirements.

Introduction

The analysis of mitochondrial DNA (mtDNA) has become an important molecular genetic tool in forensic genetics. This molecule is much more abundant in cells than nuclear DNA (nDNA), lending to its successful typing in samples that do not contain enough nDNA [1,2]. Also, mtDNA seems to be less sensitive to degradation in compromised samples, which are commonly observed in forensic work [3]. Finally, mtDNA is inherited along the maternal line, which enables the identification of distant maternally-related individuals (e.g. [4]) and the estimation of the biogeographic origin of mtDNA lineages [5], which are both relevant applications in forensic genetics.

Traditionally, the control region (CR) or its hypervariable segments (HVS-I, HVS-II, HVS-III) have been sequenced using Sanger-type sequencing (STS, [1,2,6]). With the emergence of massively parallel sequencing (MPS) techniques, a wider variety of applications has become available to the forensic genetics community including capture-based library preparation methods [[7], [8], [9]] and full mitochondrial genome (mitogenome) sequencing [[10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]]. The move towards MPS comes with increased support of typing kits and software solutions by commercial suppliers compared to STS-based mtDNA analysis, and these amplification and library generation kits have been developed and tested by the forensic genetics community to improve performance of mitogenome MPS in recent years [[10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]].

Here, we evaluated the Precision ID mtDNA Whole Genome Panel (PID mtDNA WG Panel; Thermo Fisher Scientific, Waltham, MA) by analysing more than 500 mitogenomes from diverse phylogenetic backgrounds and different forensically relevant tissues. We present and discuss the general performance of the kit and the analysis methods, concordance with previously generated STS and MPS (Illumina) data of a subset of the samples (Table S1), heteroplasmy detection, and haplogrouping of the resultant sequences.